Cellulose chromatography support

ABSTRACT

This invention relates to an improved cellulose chromatography support and, in particular, to substantially spherical, high density cellulose particles. This invention also relates to a method of making these spherical, high density cellulose particles and, in particular, to a method for forming spherical cellulose from a high molecular weight viscose in a stable emulsion of a liquid carrier and emulsifying agents.

This application is the national phase of PCT International ApplicationNo. U.S. 90/03716, filed Jun. 29, 1990, which is a continuation-in-partof U.S. application Ser. No. 07/374,281, filed Jun. 30, 1989, nowabandoned.

This invention relates to an improved uncrosslinked cellulosechromatography support and, in particular, to a support comprisingsubstantially spherical, high density cellulose particles. Thisinvention also relates to a method of making such substantiallyspherical, high density cellulose particles and, in particular, to amethod for making spherical cellulose particles from viscose in a stableemulsion of a liquid carrier and emulsifying agents.

BACKGROUND OF THE INVENTION

Cellulose and cellulose derivatives have been used as chromatographicsupports and as polymeric carriers. General chromatographic uses includeanalytical and preparative column chromatography, thin layerchromatography, ion exchange, gel chromatography and chelation andaffinity sorbents. In addition, cellulose particles may be used asfillers and bulking agents in pharmaceuticals, cosmetics, and foodproducts. Although natural abundance and availability, coupled with avariety of known derivatization schemes, make cellulose an attractivechromatographic support, it has generally suffered from severaldisadvantages. The most notable disadvantages are mechanical instabilityand poor flow characteristics.

Cellulose is a naturally occurring polymer made of linked glucosemonomers. In the native state, adjacent polymeric glucose chains areextensively hydrogen bonded in some regions and less hydrogen bonded inothers. The regions of relatively high hydrogen bonding are generallyreferred to as "microcrystalline regions" while the less hydrogen bondedregions are referred to as amorphous regions. For chromatographicapplications, it is generally desirable to limit the amorphous regions,and to utilize cellulose having either a fibrous or a microgranularform. Such fibrous and microgranular materials are generally prepared bylimited acid hydrolysis of bulk cellulose which results in thepreferential loss of interchain amorphous regions and increases themicrocrystalline regions. Both fibrous and microgranular cellulosecompositions are generally referred to as microcrystalline cellulose.

Procedures typically used to prepare microcrystalline cellulosegenerally result in aggregated particles which require grinding andparticle size separation to yield materials suitable for chromatographicpurposes. Further, the individual microcrystalline cellulose particlesare relatively irregularly shaped and fragile. These features adverselyeffect the use of these types of materials as chromatographic beds orcolumns because microcrystalline cellulose tends to easily clog andcompact. In addition, these materials tend to break down and generatefines when subject to elevated pressure. These drawbacks can result inunacceptable flow characteristics and poor chromatographic separations.

The use of cellulose in the form of crosslinked beads or sphericalparticles may partially overcome the poor flow characteristics ofmicrocrystalline cellulose chromatographic supports. When cellulose ismade into beads using known procedures, however, the porosity of thecellulose particles and their wide range of sizes cause the beads to besubject to mechanical breakdown when used in packed beds or columns.Although, mechanical stability may be improved by the addition of crosslinking agents, these crosslinking agents may increase the expense ofthe support, complicate the manufacturing processes and limit thegeneral applicability of use of the support.

Further, when placed in aqueous solutions, porous cellulose particlestypically swell significantly. Swelled, porous cellulose beads sufferfrom sensitivity to changing ionic strengths in eluting buffers andsolvents. As a result, conventional, swellable cellulose supports musttherefore be used within a specified narrow range of ionic strengths. Ifthis specific range of ionic strengths is exceeded, the swelledcellulose particles compact or shrink which results in very poor flowcharacteristics and leads to either poor chromatographic separation orto no separation at all.

Several methods of preparing spherical cellulose particles are known.One method for preparing cellulose beads extrudes a viscose at highspeed through a nozzle into a spinning acidic coagulating bath. Anothermethod forms a dispersion in an organic solvent with a surfactant andthen coagulates the suspension by pouring it into an acid solution.These procedures generate porous cellulose particles of variable anduncontrolled size distribution and suffer from the undesired formationof aggregates, agglomerates, and conglomerates of irregular and deformedshapes. These problems are believed due to the coagulation of thecellulose under changing hydrodynamic conditions. A third procedurethermally forms cellulose particles by heating an aqueous suspension oflow molecular weight sodium cellulose xanthate in a stirred, relativelylow viscosity, water-immiscible liquid. Although this procedure uses arelatively-constant, hydrodynamic environment for bead formation, thethermal decomposition of the sodium cellulose xanthate results in porousparticles having a wide range of sizes.

There exists a need for a spherical, high density cellulosechromatographic support which has excellent mechanical stability, whichmay be used over a wide range of pH values and ionic strengths, andwhich may be readily prepared by a reproducible general method whichprovides uniformly sized and shaped particles.

SUMMARY OF THE INVENTION

It is an object of this invention to overcome one or more of theproblems listed above.

The present invention encompasses a cellulose chromatographic supportcomprising substantially spherical, high density cellulose particleswhere the particles have a bulk dry density of about 0.65 to 0.85 g/ml,where each of the particles have an average diameter in the range ofless than about 200 microns and where the particles are essentiallynonswelling in aqueous or organic solutions. Preferably, the celluloseparticles are further characterized by i) increased stability insolutions of various ionic strengths and hydrogen bond breakingcapability, including solutions up to at least 3.0 M potassium chlorideand 8 M urea, respectively; ii) stability over a pH range from about 3to 12; iii) compatibility with both aqueous and organic solvents; iv)capability to be modified with chromatographic ligands; and v) excellentflow rates when used as a column chromatography support.

The spherical cellulose particles of the present invention may bechemically modified by covalently binding chromatographic ligands to theparticles in high loading ratios, where the loading ratio is defined asthe moles of ligand to the mass of particles. Preferred ligands includecovalently bound polar and nonpolar ligands. Suitable ligands mayinclude aminoethyl, diethylaminoethyl, epichlorohydrin triethanol amine,polyethyleneimine, methyl polyethyleneimine, benzyldiethylamine ethyl,diethyl-[2hydroxypropyl]-aminoethyl, triethylaminoethyl, sulphopropyl,carboxymethyl, sulphonate, quaternary ammonium ethyl, antigens andantibodies. A particularly preferred ligand is a polyethyleneimine whichallows the modified support to be used as an ion exchange support.

The present invention also encompasses a method of making substantiallyspherical, high density cellulose particles comprising the steps of: i)forming a stable emulsion of technical viscose in the presence of atleast one emulsifying agent and a liquid carrier, where the temperatureof said viscose during the formation of the emulsion does not thermallydecompose said viscose; ii) regenerating cellulose from said viscose inthe absence of acid over a period of time, with continuous agitationunder stationary hydrodynamic conditions, to yield a dispersion ofuniformly sized, deformable particles; iii) contacting said dispersionof deformable particles with a solvent suitable to cause a partialextraction of said liquid carrier from said emulsion, wherein saiddeformable particles begin to partially harden, and; iv) hardening thedeformable particles. The preferred temperature for formulating theemulsion and regenerating the cellulose is less than 30° C., and mostpreferably at temperatures of about 20 to 30° C. The regeneration ofcellulose from viscose preferably extends over a period of about six toeighteen hours. A suitable liquid carrier has a viscosity greater thanabout 100 cSt at ambient temperatures.

Preferred high viscosity liquid carriers have viscosities greater than150 cSt as measured using standard techniques at ambient temperatures;the most preferred carrier is polypropylene glycol having an averagemolecular weight of about 1200 and a viscosity of about 160 cSt attemperatures of about 20 to 30° C.

The spherical particles may be further hardened by stirring thepartially hardened particles in an acidic alcoholic solution preferablya solution comprising 30% acetic acid in ethanol, a solution comprising30% propanoic acid in propanol, or a solution comprising 20% phosphoricacid in ethanol.

Other objects and advantages of this invention will be apparent uponconsideration of the following detailed description which includesillustrative examples of the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photomicrograph (magnification 500×) of spherical celluloseparticles having a diameter of between about 25 to 45 microns. FIG. 2 isa photomicrograph (magnification 2000×) of a single cellulose particlethat was mechanically cut in half. FIG. 3 is also a SEM photomicrographof a cellulose particle imbedded in a polymer film that was also cut inhalf (magnification 2000×).

DETAILED DESCRIPTION

This invention relates to a cellulose chromatographic support comprisingsubstantially spherical, high density cellulose particles where theparticles of the invention have a bulk dry density of about 0.65 to 0.85g/ml, where each of the particles have an average diameter of less thanabout 200 microns, and where the particles are essentially nonswellingin aqueous or organic solutions.

The phrase "essentially nonswelling," as used herein, refers tocellulose particles which do not appreciably change in volume whencontacted with either aqueous or organic solvents or solutions. That is,the cellulose particles of the present invention are resistant to bothswelling and contracting when placed in a variety of organic solvents ofdifferent dielectric constants or polarities. No appreciable change involume occurs in nonpolar solvents such as hexane, toluene, and benzeneor in polar solvents such as water, dimethylsulfoxide,dimethylformamide, or N-methyl pyrrolidone. In addition, hydrogen bondbreaking solvents, such as 8 M urea, do not result in any volume change.This property may be contrasted with known cellulose beads which swellto a large extent when placed in water or other solvents. It is believedthat the volume retention properties of the spherical particles of thisinvention are related to the relatively high density and thecorrespondingly low porosity of the particles. For example, 10 to 25micron particles have a bulk dry apparent density of 0.8045 g/ml; 25 to53 micron particles have an apparent density of 0.7923 g/ml; and 53 to177 micron particles have an apparent density of 0.6789 g/ml.

As illustrated by FIGS. 2 and 3 the particles of the present inventionhave very low porosity. The particles are substantially solid whenviewed under a microscope or by SEM a at magnification of 2000X. Thephotomicrographs illustrated in FIGS. 1 and 2 were taken on a JEOL ModelJSM-354 scanning electron microscope. The photomicrograph illustrated inFIG. 3 was taken on a JEOL Model 840 scanning electron microscope. Thecellulose particle shown in the photomicrograph was imbedded in acollodion polymer film and then cut in half.

The cellulose particles may be chemically modified to covalently bindchromatographic ligands to the particles. Suitable ligands include anyof a variety of known polar or nonpolar ligands. Polar ligands may beeither cationic or anionic compounds and nonpolar ligands may beuncharged or neutral compounds. In particular, covalently boundpolyethyleneimines allow the modified supports to be used as ionexchange supports. Other known ligands may give useful supports foraffinity, reverse phase and hydrophobic chromatography. Suitable ligandsmay include aminoethyl, diethylaminoethyl, epichlorohydrintriethanolamine, polyethyleneimine, methyl polyethyleneimine,benzyldiethylamine ethyl, diethyl-[2-hydroxy propyl]-aminoethyl,triethylaminoethyl, sulphopropyl, carboxymethyl, sulphonate, quaternaryammonium ethyl, antigens and antibodies.

Known chromatographic ligands can be covalently bound to the hydroxylgroups on the accessible surfaces of the particles using difunctionallinking molecules which have two reactive sites associated with eachmolecule. Any difunctional linking molecule known to react with hydroxylgroups of cellulose may be used. Particularly preferred linking groupswith two reactive sites include epichlorohydrin, benzoquinone, anddiglycidyl ethers of alkane diols. In performing this binding step, thelinking group is typically contacted with the cellulose particles underconditions suitable to covalently bind to the cellulose using onereactive site on the linking molecule. The resulting intermediate isthen contacted with a known, reactive chromatographic ligand which bindsto the second reactive site of the linking group. Suitable reactivechromatographic ligands are any of a variety of ligands well known tothose skilled in the art. A preferred ligand used to form an ionexchange chromatographic support is a reactive polyethyleneimine.

The cellulose particles of this invention possess several desiredcharacteristics. For example, the cellulose particles of this inventionare mechanically stable. No mechanical breakdown of the particlesoccurred under intense magnetic stirring in various organic solvents orin acidic aqueous solutions for time periods of between five and twelvehours.

The cellulose particles are also stable to elevated pressures when usedin chromatographic columns. The spherical cellulose particles provideexcellent flow rates over a range of pressures and conditions. Forexample, when the cellulose particles were packed in a column andsubjected to medium pressure conditions provided by a fluid meteringpump there was no mechanical breakdown at pressures up to at least 120psi for extended periods of time and there was no appreciable decreasein the flow rates or in the volume of the chromatographic bed as thepressure was increased.

The method for making substantially spherical, high density celluloseparticles of this invention comprises the steps of: i) forming a stableemulsion of technical viscose in a liquid carrier without thermallydecomposing the viscose; ii) regenerating the cellulose to givedeformable spherical particles; and, iii) further hardening thedeformable particles.

The phrase, "technical viscose," as used herein, means an aqueoussolution generated from pulp containing high molecular weight cellulose,sodium hydroxide, and sulfur which results from the substitution ofhydroxyl groups with xanthate groups. A suitable viscose may containabout 7.0% high molecular weight cellulose, about 5.0% sodium hydroxideand about 1.7% sulfur. This viscose may have about 20% of the hydroxylgroups of the cellulose substituted with xanthate groups. A descriptionof the process to generate suitable viscose from wood pulp is found inU.S. Pat. No. 4,778,639.

The phrase, "liquid carrier," as used herein, means a liquid solventpreferably having a viscosity greater than 100 cSt at ambienttemperatures and more preferably greater than about 150 cSt attemperatures of about 20 to 30° C. which is capable of forming a stableemulsion with viscose in the presence of emulsifying agents. The phrase"stable emulsion", as used herein, means an emulsion of viscose formedin the liquid carrier using emulsifying agents such that the emulsiondoes not break down during the time period required to regeneratecellulose from viscose A stable emulsion is preferred because theregeneration of cellulose from viscose may be allowed to occur in acontrolled manner. If the regeneration is uncontrolled or if thegeneration of carbon disulfide during the regeneration is too rapid avery porous product is formed. In addition, if the emulsion breaks downduring the regeneration period, the developing particles are likely tolose the desired spherical shape and are likely to form undesiredirregularly shaped aggregates and agglomerates.

It was empirically observed that the combination of low viscositysolvents and high molecular weight viscose typically did not generatestable emulsions. For example, emulsifying agents and relatively lowviscosity solvents such as dichlorobenzene, chloroform, chloroform andparaffin, paraffin, hexane, cyclohexane, silicone oil, mineral oil, andtoluene all failed to provide stable emulsions with high molecularweight viscose. In general, the inability to generate stable emulsionsleads to large unusable clumps of partially dexanthated celluloseinstead of spherical cellulose particles.

It was also observed that as the viscosity of the solvent increased, thestability of the emulsion increased. For example, Arcoprime, (viscosityabout 78.5 cSt), generated a relatively poor emulsion but yieldedparticles of irregular sizes and shapes. High viscosity solvents such aspolypropylene glycol 1200 (viscosity about 160 cSt), Polymeg 650(viscosity about 650 cSt) and Cargille type B immersion oil (viscosityabout 1250 cSt) yielded stable emulsions when used in conjunction withemulsifying agents and also resulted in satisfactory sphericalparticles. A preferred solvent is polypropylene glycol 1200.

The emulsifying agents may be optimized to vary the stability of theemulsions. Preferred emulsifying agents, used in combination withpolypropylene glycol having an average molecular weight of about 1200,include sorbitan monooleate and Tween-80® (polyoxyethylene (20) sorbitanmonooleate) and Trylox CO-5®. A preferred weight percent ratio ofsorbitan monooleate to Tween-80® in polypropylene glycol is about 75-98wt.% sorbitan monooleate and 2-25 wt.% Tween-80®. Particularly preferredweight percent ratios of sorbitan monooleate to Tween-80® inpolypropylene glycol are 80-20 wt.% and 96.5-3.5 wt.% respectively. Apreferred weight percent ratio of sorbitan monooleate to Trylox CO-5® inpolypropylene glycol is about 9-30 wt.% sorbitan monooleate and 70-91wt.% Trylox CO-5®. A particularly preferred weight percent ratio is9.724 wt.% sorbitan monooleate and 90.276 wt.% Trylox CO-5®.

A suitable test to determine an optimized emulsion system may be readilyperformed by making up a a sample mixture by adding technical viscose(0.3 ml) to polyethylene glycol having a average molecular weight ofabout 1200 (1.5 ml) that had been previously vortexed with varyingweight percentages of emulsifiers (0.1 ml). The sample mixture isvortexed for one minute, and the mixture is used to fill Wintrobeerythrocyte sedimentation tubes. Two tubes for each mixture are used andthe tubes are then centrifuges for 5 minutes. A ratio is taken betweenthe amount of viscose still emulsified after centrifugation and thetotal volume of fluid in the sedimentation tube. The emulsions with thehighest ratios of the amount of emulsified viscose to the total fluidvolume are considered to have the optimum emulsifier concentrations. Theemulsion system test is typically run in duplicate and the results areaveraged. Small differences in viscose temperature, room temperature, orexact time of centrifugation are corrected for by using the an optimumconcentration from an earlier run as control.

Another method to determine the stability of a particular emulsionsystem may be performed by a visualization test. To perform the test, adrop of a viscose emulsion to be visualized is placed on a clean glassslide, and another slide is placed on top of this drop, slightly offset.The bottom slide is then held at a downward angle, allowing the top oneto slowly slide down, resulting in one layer of emulsion droplets on theoriginal slide. This slide is then allowed to dry in a verticalposition. After drying for about 2 days, the slide is placed in a coplinjar filled with acetone for about 1 or 2 days in order to remove theremaining solvent. After removal of the solvent from the slide, theslide is viewed directly under a microscope under low power, whichallows visualization of the size of the emulsified viscose at the timethe emulsion is forming.

Using these simple tests it is possible to define the weight percentconcentration of the emulsifies in the liquid carrier to about onethousandth of a percent. This accuracy is preferred in order to achievean optimized emulsion system to practice the method of this invention.

When a technical viscose is used to make cellulose particles accordingto the present invention, a salt solution is preferably added to theviscose. The resulting spherical particles are obtained and thesespheres are smaller than the particles obtained using the same processbut which are formed without adding a salt solution to the viscoseemulsion. Typical salt solution may include aqueous potassium chloridesolutions.

Another method for producing smaller spheres is to increase blendingtime when forming the viscose emulsion.

The formation of desired spherical cellulose particles in the absence ofacid is also dependent on the temperature used to form the emulsion andthe temperature used to regenerate the cellulose from the viscose. Attemperatures above room temperature, as the temperature is increased,the range of the size distribution of the particles is generallyincreased, the particles are more susceptible to forming aggregates andclumps and the particles may be softer and more porous depending onambient pressure. Preferably, when practicing the method of thisinvention at atmospheric pressure, the temperature is maintained belowabout 30° C; the most preferred emulsifying and regeneratingtemperatures are in the range of about 20 to 30° C. Increasing theambient pressure and lowering the temperature below about 20° C. mayproduce particles with reduced porosity; however, the regeneration timeis increased and the production rate is reduced accordingly. It has alsobeen observed that the final temperature of the emulsion is lower whenthe solvents used are stored under refrigeration and the temperature ofthe blender cooling bath is low.

The particles obtained after regeneration are relatively soft and may beeasily deformed if mishandled. Therefore, the regenerated celluloseparticles are preferably suspended in a nonpolar solvent such as hexaneor toluene, in order to extract the liquid carrier and to initiateparticle hardening. Further hardening occurs by stirring the particlesin an acidic alcoholic solution, such as 30% acetic acid in ethanol, 30%propanoic acid in propanol, or 20% phosphoric acid in ethanol.Preferably, the hardening of the particles in an acidic alcoholicsolution is done at low temperatures in the range of about -30° to 30°C.

As a final step in the process, the hardening of the particles may beaccelerated by contacting the particles with stepped gradients ofdimethylsulfoxide and t-butylmethyl ether, dimethylsulfoxide and diethylether, or dimethylsulfoxide and propanol solutions at temperatures ofabout -30° to 30° C.; at higher temperatures, the reaction is faster, atlower temperatures, the resulting particles are more uniform.

The substantially spherical, high density cellulose particles of thisinvention are useful in a variety of applications. When the celluloseparticle is modified with a polar chromatographic ligand, the materialmay be used as a high performance, thin layer chromatographic sorbent toseparate biological materials such as nucleic acids, oligonucleotides orrelated compounds. The same modified material may be used in a smallcolumn as an ion exchange sorbent to purify biomolecules. When thecellulose particle is modified with a nonpolar chromatographic ligand,the material may be used in solid phase extractions such as inanalytical drug testing procedures or in extracting difficult-to-removeorganic materials from aqueous solutions. In the unmodified state, theexceptional physical characteristics of the cellulose particles of thisinvention allow the particles to be used as bulking agents or fillers incosmetic, pharmaceutical or food products.

Several examples describing various embodiments of the invention aregiven below. These examples are for illustrative purposes only and arenot intended to limit the scope of the invention.

EXAMPLE 1

Cargille Type B immersion oil (200 ml, viscosity 1250 cSt) was blendedbriefly with 80 wt.% sorbitan monooleate and 20 wt.% Tween-80® (1.064 g,total weight of surfactants) and added to a solution of technicalviscose (33.5 ml) diluted with water (33.5 ml). The mixture was blended,utilizing the highest speed of a Hamilton Beach blender for two minutes,heated at a constant temperature of 38° C. and stirred overnight at aspeed of 377 rpm. Cyclohexane (100 ml) was added to the reactionmixture; the resulting particles were filtered, and washed with ethanol.The particulate matter was then stirred in 40% acetic acid in ethanol.After stirring for one hour, the particles were washed with 1-propanoland then soaked in mixtures of solvents, described below, for a periodof up to one hour per solvent mixture. The first solvent used was 100%dimethylsulfoxide (DMSO), followed by solutions of 75% DMSO/25%t-butylmethyl ether (TBME), 50% DMSO/50% TBME, 25% DMSO/ 75% TBME, and100% TBME. After the last wash with TBME, the beads were suction driedon a glass filter funnel. The following size distribution was observedafter dry sieving: 53-177 microns-90%; 25-53 microns-10%.

EXAMPLE 2

Technical viscose (33.5 ml) was added to water (33.5 ml), and stirreduntil a homogenous viscose solution was obtained. Arcoprime 350® (200ml, viscosity 78.9 cSt) was blended for 10-15 seconds with 80 wt.%sorbitan monooleate and 20 wt.% Tween-80® (1.064 g, total weight ofsurfactants) in a separate flask and the viscose solution was added toit. The resultant mixture was blended for two minutes at high speed,transferred to a reaction vessel and stirred at 400 rpm overnight at aconstant temperature of 39° C. The resulting particles were washed andsoaked in mixtures of solvents in the same manner as described inExample 1. The following size distribution was observed upon drysieving: 53-177 microns-61%; 25-53 microns-29%; over 177 microns-10%.

EXAMPLE 3

Technical viscose (35 ml) was added to water (35 ml), and the resultingsolution was added to polypropylene glycol (PPG) 1200 (200 ml, MW 1200,viscosity 160 cSt), which had been blended with an 80 wt.% sorbitanmonooleate and 20 wt.% Tween-80® (1.064 g, total weight of surfactants).This mixture was blended for 30 seconds, blending was stopped for 15seconds, and the same sequence was repeated four times for a totalblending time of two minutes. The mixture was transferred to a reactionvessel and stirred overnight at 330 rpm at a constant temperature of 41°C. The resulting particles were centrifuged at 2500 rpm at 21° C., fortwo and one half minutes. The liquid phase was decanted from theparticles, which were stirred in hexane and decanted. The particles wereagain stirred with hexane and decanted and then stirred with ethanol.The particles were filtered on a glass filter funnel, and treated with30% acetic acid in ethanol. The resulting particles were soaked inmixtures of solvents and treated in the same manner as described inExample 1, except that ethanol was substituted for TBME. Finally, theparticles were stirred in distilled water, filtered through a glassfilter, and air dried. A qualitative molybdate test for the presence ofsulfur and an elemental analysis for sulphur were both negative. Thefollowing size distribution was observed after dry sieving: 53-177microns-97%; 25-53 microns-3%.

EXAMPLE 4

Technical viscose (100 ml) was added to water (100 ml), and the solutionwas added to PPG 1200 (600 ml), which had been blended with 80 wt.%sorbitan monooleate and 20 wt.% Tween-80® (1.064 g, total weight ofsurfactants). The experiment was carried out in the same manner asExample 3, except the reaction temperature was kept at 23° C. whilestirring overnight. The following size distribution was observed afterdry sieving: 53-177 microns-67%; 25-53 microns-32%; 10-25 microns-1%.

EXAMPLE 5

PPG 1200 (550 ml) was blended with 80 wt.% sorbitan monooleate and 20wt.% Tween-80® (1.064 g, total weight surfactants) and added totechnical viscose (100 ml, undiluted). The process was continued in thesame manner as in Example 3, except that the reaction temperature was24° C. The following size distribution was observed after dry sieving:53-177 microns-97%; 25-53 microns-3%.

EXAMPLE 6

PPG 1200 (600 ml) was blended with 80 wt.% sorbitan monooleate and 20wt.% Tween-80® (1.064 g, total weight of surfactants) and added totechnical viscose (100 ml, undiluted). The mixture was emulsified athigh pressure using a piston-type fluidizer and stirred in a reactionvessel overnight at 23° C. at 375 rpm. The process was continued in thesame manner as in Example 3. The following size distribution wasobserved after dry sieving: 53-177- microns-82%; 25-53 microns-18%.

EXAMPLE 7

PPG 1200 (300 ml) was blended with 80 wt.% sorbitan monooleate and 20wt.% Tween-80® (1.064 g, total weight of surfactants) and added totechnical viscose (100 ml). The mixture was stirred in a reaction vesselat 500 rpm at a constant temperature of 22° C. The process was thencontinued in the same manner as in Example 3. The following sizedistribution was observed after dry sieving: 53-177- microns-82%; 25-53microns-5%; over 177 microns-13%.

EXAMPLE 8

PPG 1200 (600 ml) was blended with 80 wt.% sorbitan monooleate and 20wt.% Tween-80® (3.18 g, total weight of surfactants) and added to asolution of technical viscose (100.5 ml) and water (100.5 ml). Themixture was blended using a Waring Commercial Blender equipped with acooling jacket and a cooling coil (temperature of circulating water,-10° C.) for four minutes at high speed. The mixture was stirredovernight at 200 rpm at 24° C. The process was then continued in thesame manner as in Example 3. The following size distribution wasobtained by dry sieving: 53-177- microns-11%; 25-53 microns-77%; 10-25microns-8%; over 177 microns-4%.

EXAMPLE 9

PPG 1200 (600ml) was blended with an emulsifier (24 ml, 96.5 wt.%sorbitan monooleate and 3.5 wt.% Tween-80®) and added to undilutedviscose (200 ml). The process was then continued in the same manner asdescribed in Example 8. The following size distribution was observedafter dry sieving: 53-177 microns-72%; 25-53 microns-28%.

EXAMPLE 10

Polymeg 650®(polytetramethylene ether glycol, 300 ml) was blended withan emulsifier (12ml, 96.5% sorbitan monooleate and 3.5% Tween-80®) andadded to undiluted viscose (100 ml). The mixture was blended using aWaring Commercial Blender equipped with a cooling jacket and a coolingcoil (temperature of circulating water -10° C.) for 7 minutes at highspeed. The mixture was stirred at 628 rpm overnight at 24° C. Aftercentrifuging at 2500 rpm for 2.5 minutes at 21° C., the liquid fractionwas decanted and discarded. The particles were twice stirred in propanoland decanted. The particles were filtered and added to a solution of 20%phosphoric acid in ethanol. The particles were washed following theprocedure describe in Example 3. The following size distribution wasobserved after dry sieving: 53-177 microns-98%; 25-53 microns-2%.

EXAMPLE 11

Technical viscose (100 ml) was diluted with a solution of 1 M potassiumchloride (100 ml) and the resulting solution was added to a blendedsolution of PPG 1200 (500 ml), 95.9 wt.% sorbitan monooleate and 4.1wt.% Tween-80® (22 ml total volume of surfactants). The formation ofparticles was carried out according the procedure described in Example 8above. The following size distribution was observed by dry sieving:25-53 microns-80%; 10-25 microns-20%. Microscopic examination showed amuch higher percentage of particles in the 10-25 micron range with manyparticles having a size of less than 10 microns, but the low mass ofthese particles hindered separation using conventional sieves.

EXAMPLE 12

Technical viscose (100 ml) was diluted with an aqueous potassiumchloride solution (5.56g KCl in 25 ml water) resulting in concentrationof salt in the diluted viscose solution of about 1 M. The resultingsolution was added to a blended solution of PPG 1200 (275 ml), 95.9 wt.%sorbitan monooleate and 4.1 wt.% Tween-80® (12 ml total volume ofsurfactants). The mixture was blended using a Waring Commercial Blenderfor 10 minutes on speed #6, followed by 2 minutes on speed #7 in thesame manner as Experiment 8 and the formation of particles was carriedout according to the procedure described in example 4 above. Thefollowing size distribution was observed by dry sieving: 25-53microns-78%; 10-25 microns-22%. Microscopic examination showed a muchhigher percentage of the particles in 10-25 micron range in contrast tothe distribution achieved through sieving, with many particles having asize of less than 10 microns, but the low mass of these particlesprevented separation using conventional sieves.

EXAMPLE 13

Technical viscose (120 ml) was added to a blended solution of PPG 1200(300 ml) 9.724 wt.% sorbitan monooleate and 90.276 wt.% Trylox CO-5® (22ml total volume of surfactants, Trylox CO-5® manufactured by Emery was acastor oil ethoxylate containing 5 moles ethylene oxide per mole ofcastor oil). This mixture was blended for a total of 3 minutes on lowspeed, using a Waring Commercial Blender in the same manner as Example 8and the formation of particles was carried out according to theprocedures described as Example 4 above. The following size distributionwas observed by dry sieving: 38-53 microns-52%; 25-38 microns 37%; 10-25microns 11%.

EXAMPLE 14

Technical viscose (600 ml) was added to a blended solution of PPG 1200(1500 ml), 30.0 wt.% sorbitan monooleate and 70.0 wt.% Trylox CO-5® (110ml total volume of surfactants). This mixture was blended for a total of3 minutes on low speed using a Waring Commercial Blender in the samemanner as Example 8 and the formation of particles was carried outaccording to the procedures described in Example 4. The following sizedistribution was observed by dry sieving: 38-53 microns-86%; 25-38microns-14%. The particles were mainly individual spheres, althoughthere were also some clusters of spheres.

EXAMPLE 15

Technical viscose (125 ml) was added to a blended solution of PPG 1200(250 ml) 0.725 wt.% sorbitan monooleate and 90.275 wt.% Trylox CO-5® (15ml total volume of surfactants). This mixture was blended for a total of15 minutes on medium speed using a Waring Commercial Blender in the samemanner as Example 8 and the formation of particles was carried outaccording to the procedures described in Example 4 above. The followingsize distribution was observed by dry sieving: 38-53 microns-11%; 25-38microns-82%; 10-25 microns-7%.

EXAMPLE 16

Cellulose particles (38-53 microns) were packed in a 8.5×0.8 cmMichel-Miller column using the slurry technique as described in the AceMichel-Miller brochure. The column was packed at about 120 psi backpressure using a fluid metering pump. Solvent (water or a 2M KClsolution) was pumped through the bed at back pressures of about 100 psiover a period of 64 hours. Flow rates of 370 ml per hour were recordedat stable bed volume. Examination of the cellulose particles under themicroscope (100×) showed no visible deformation of the particles.

Similarly, cellulose particles (38-53) microns) were dry packed in a8.5×0.8 cm Michel-Miller column which was then connected to a fluidmetering pump operating at about 40 psi back pressure. The bed volumeincreased upon wetting by about 6%. A volume of 5 liters of 2M KCl waspassed through the column. The bed volume remained constant throughoutthe experiment.

EXAMPLE 17

Cellulose particles (20-38 microns) were allowed to soak in water for 2hours, after which they were filtered on a fritted glass filter, allowedto air dry overnight at room temperature and then weighed (2.9917 g).The air dried particles were then dried in a vacuum dessicator for aperiod of 24 hours at reduced pressure (10⁻³ mm Hg) and reweighed (finalweight 2.7799 g). The weight lost due to remaining water absorbed on theparticles was about 7%.

EXAMPLE 18 ATTACHMENT OF PEI TO CELLULOSE WITH BENZOQUINONE

Polyethyleneimine (PEI) was attached to cellulose using benzoquinone inthe following manner. Cellulose (0.4117 g) was placed in contact with anacetate buffer (0.1 M, pH 5.4) for one hour and filtered. Fifty (50) mlof a solution prepared by dissolving Benzoquinone (1.6215 g) in asolution of ethanol (60 ml) and acetate buffer (240 ml) was added to thecellulose filtrate and the reaction vessel was rotated gently for onehour. The cellulose was filtered and washed with 0.5 M sodium chlorideand acetate buffer until the wash was unreactive to base. A solution ofPEI₁₈ (111.3 mg) in ethanol (100 ml) was then added to the wetcellulose. The mixture was protected from light and the reaction vesselwas rotated gently overnight. The resulting solid was washed withacetate buffer (500 ml), sodium chloride (500 ml), and methanol (500ml). Acetic acid (20%) in acetate buffer was added to the solid, themixture was vortexed, centrifuged, decanted and blotted. This sequencewas repeated four times. The particles were qualitatively tested for thepresence of amines and the results of the test were positive, indicatingthe covalent attachment of PEI to the cellulose particle.

EXAMPLE 19 ATTACHMENT OF PEI TO CELLULOSE WITH EPICHLOROHYDRIN

PEI was attached to cellulose using epichlorohydrin in the followingmanner. Cellulose particles (1.0 g) were equilibrated with acetatebuffer (0.1 M, pH 5.4) for one hour and filtered. A sodium borohydridesolution (5 ml, made by mixing sodium hydroxide (500 ml, 2N), sodiumborohydride (2.5 g) and diluting the mixture 1:2 with water) was addedto the equilibrated cellulose particles. Epichlorohydrin (1.0 ml) wasadded to the mixture and the reaction vessel was rotated gently at 60°C. for one hour. The mixture was washed with warm water (60° C.), andmixed with a PEI18 solution (4 ml, 37 mg PEI/ml), and the reactionvessel was rotated for an additional 30 minutes at 60° C. The productwas then washed with hot brine. Elemental analysis: 3.03% nitrogen,corresponding to 96.06 mg PEI bound per 1.0 g cellulose.

EXAMPLE 20 ATTACHMENT OF PEI TO CELLULOSE WITH A DIGLYCIDYL ETHER

A solution (1 ml, sodium hydroxide (0.6 M) containing sodiumborohydride, 2 mg) was added to 1,4-butanediol diglycidyl ether (1.0ml). The mixture was added to cellulose (1.0 g) and the reaction vesselwas rotated for seven hours at 30° C. The cellulose was washed withwater and added to a solution (4 ml) containing PEI₁₈ (148.78 mg). Thereaction vessel was rotated at 35° C. for 30 minutes and the product waswashed with warm water (80-90° C.) and aqueous NaCl/NaOH (1 M).Elemental analysis: 3.60% nitrogen, corresponding to 110.57 mg PEI boundper 1.0 g cellulose.

We claim:
 1. A chromatographic support comprising substantiallyspherical, non-cross-linked, high density cellulose particles whereinsaid particles have a bulk dry apparent density of about 0.65 to 0.85g/ml, wherein said particles have an average diameter in the range offrom about 25 microns to about 200 microns, and wherein said particlesare essentially nonswelling in aqueous or organic solutions.
 2. Thecellulose particles according to claim 1 wherein said particles arestable in aqueous solutions of up to at least 3.0 M potassium chlorideand 8 M urea.
 3. The cellulose particles according to claim 1 furthercomprising chromatographic ligands covalently bound to said particles.4. The cellulose particles according to claim 3 comprising polarchromatographic ligands covalently bound to said particles.
 5. Thecellulose particles according to claim 3 comprising nonpolarchromatographic ligands covalently bound to said particles.
 6. Thecellulose particles according to claim 4 wherein said covalently boundligands are selected from the group consisting of aminoethyl,diethylaminoethyl, epichlorohydrin triethanolamine, polyethyleneimines,methyl polyethyleneimines, benzyl diethylamine ether,diethyl-[2-hydroxypropyl]aminoethyl, triethylaminoethyl, sulphopropyl,carboxymethyl, sulphonate, quaternary ammonium ethyl, antigens andantibodies.
 7. The cellulose particles according to claim 1 wherein saidparticles are stable at pressures up to 120 psi.
 8. The celluloseparticles according to claim 1 wherein said particles are mechanicallystable.
 9. A method of making substantially spherical, high densitycellulose particles comprising the steps of:forming a stable emulsion ofviscose in the presence of at least one emulsifying agent and a liquidcarrier, wherein the temperature of said viscose during the formation ofsaid emulsion does not thermally decompose said viscose; regeneratingcellulose from said viscose in the absence of acid over a period of timewith continuous agitation under stationary hydrodynamic conditions toyield a dispersion of uniformly sized, deformable particles; contactingsaid dispersion of deformable particles with a solvent suitable to causea partial extraction of said liquid carrier from said emulsion, whereinsaid deformable particles begin to partially harden; and hardening saiddeformable particles whereby high density particles having an averagediameter in the range of from about 25 microns to about 200 microns areproduced.
 10. The method according to claim 9 wherein said temperatureis above the freezing point of said emulsion, but less than 30°.
 11. Themethod according to claim 10 wherein said temperature is about 20° to30° C.
 12. The method according to claim 9 wherein said liquid carrierhas a viscosity greater than 150 cSt at temperatures of about 20° to 30°C.
 13. The method according to claim 12 wherein said liquid carrier ispolypropylene glycol having an average molecular weight of about 1200and a viscosity of about 160 cSt at temperatures of about 20° to 30° C.14. The method according to claim 9 wherein said emulsion is formed inthe presence of at least two emulsifying agents.
 15. The methodaccording to claim 14 wherein said emulsifying agents are neutralsurfactants selected from the group consisting of sorbitan,polyoxyethylene (20) sorbitan monooleate and a castor oil ethoxylate.16. The method according to claim 9 wherein said particle hardeningcomprises stirring said deformable particles in an acidic alcoholicsolution.
 17. The method according to claim 9 further comprising addinga salt solution to the viscose emulsion.
 18. Spherical, high densitycellulose particles made by a method comprising the steps of:forming astable emulsion of viscose in the presence of at least one emulsifyingagent and a liquid carrier, wherein the temperature of said viscoseduring the formation of said emulsion does not thermally decompose saidviscose; regenerating cellulose from said viscose in the absence of acidover a period of time with continuous agitation under stationaryhydrodynamic conditions to yield a dispersion of uniformly sized,deformable particles; contacting said dispersion of deformable particleswith a solvent suitable to cause a partial extraction of said liquidcarrier from said emulsion, wherein said deformable particles begin topartially harden; and hardening said deformable particles to producehigh density particles having an average diameter in the range of fromabout 25 microns to about 200 microns.